A novel clot lysis assay for recombinant plasminogen activator.

Recombinant plasminogen activator (r-PA, reteplase) is an engineered variant of alteplase. When expressed in E. coli, it appears as inclusion bodies that require refolding to recover its biological activity. An important step following refolding is to determine the activity of refolded protein. Current methods for enzymatic activity of thrombolytic drugs are costly and complex. Here a straightforward and low-cost clot lysis assay was developed. It quantitatively measures the activity of the commercial reteplase and is also capable of screening refolding conditions. As evidence for adequate accuracy and sensitivity of the current assay, r-PA activity measurements are shown to be comparable to those obtained from chromogenic substrate assay.

Effect of arginine on pre-nucleus stage of interferon beta-1b aggregation.

Understanding the mechanism of aggregation of a therapeutic protein would not only ease the manufacturing processing but could also lead to a more stable finished product. Aggregation of recombinant interferon (IFNß-1b) was studied by heating, oxidizing, or seeding of unformulated monomeric solution. The formation of aggregates was monitored by dynamic light scattering (DLS) and UV spectroscopy. The autocatalytic monomer loss model was used to fit the data on aggregation rates. The influence of pre-nucleation on aggregation step was demonstrated by inducing the liquid samples containing a monomer form of folded IFNß-1b by heat and also an oxidizing agent. Results tend to suggest that the nucleus includes a single protein molecule which has been probably deformed. Seeding tests showed that aggregation of IFNß-1b was probably initiated when 1.0% (w/w) of monomers converted to nucleus form. Chemiluminescence spectroscopy analysis of the sample indicated the generation of 3.0 µM of hydrogen peroxide (H2O2) during nucleation stage of IFNß-1b aggregation. Arginine with a concentration of 200 mM was sufficient to suppress aggregation of IFNß-1b by decreasing the rate of pre-nucleation step. We proposed the formation of pre-nucleus structures prior to nucleation as the mechanism of aggregation of IFNß-1b. Furthermore, we have showed the positive anti-aggregation effect of arginine on pre-nucleation step.

Polysaccharides and proteins-based bionanocomposites for microencapsulation of probiotics to improve stability and viability in the gastrointestinal tract: A review.

Probiotics have recently received significant attention due to their various benefits, such as the modulation of gut flora, reduction of blood sugar and insulin resistance, prevention and treatment of digestive disorders, and strengthening of the immune system. One of the major issues concerning probiotics is the maintenance of their viability in the presence of digestive conditions and extended shelf life during storage. To address this concern, numerous techniques have been explored to achieve success. Among these methods, the microencapsulation of probiotics has been proposed as the most effective way to overcome this challenge. The combination of nanomaterials with biopolymer coating is considered a novel approach to improve its viability and effective delivery. The use of polysaccharides and proteins-based bionanocomposites for microencapsulation of probiotics has emerged as an efficient and promising approach for maintaining cell viability and targeted delivery. This review article aims to investigate the use of different bionanocomposites in microencapsulation of probiotics and their effect on cell survival in long-term storage and harsh conditions in the gastrointestinal tract.

FTIR Investigation of Secondary Structure of Reteplase Inclusion Bodies Produced in Escherichia coli in Terms of Urea Concentration.

Recent studies suggest that reducing the induction temperature would improve the quality of some recombinant inclusion bodies (IB) by providing a native-like secondary structure and leading to an improvement in protein recovery. This study focused on optimizing the solubilization condition of Reteplase, a recombinant protein with 9 disulfide bonds. The influence of lowering induction temperature and urea concentration was investigated on the secondary structure of the recombinant protein through FTIR analysis. Induction temperature reduction decreased the percentage of helixes and loops from 49 to 8. In addition, FTIR spectroscopy corroborates the drastic impact of this parameter on Reteplase secondary structure. Even though lowering urea concentration tripled the solubility of IBs expressed at lower induction temperature, the final yield is still quite low to be considered as optimum. On the other hand, the percentage of beta strands and turns in secondary structure of dissolved proteins were proportional to urea concentration. Therefore, in case of Reteplase, protein expression at low temperature (25 °C) was not efficient to improve the protein recovery yield. Future studies need to focus on using other methods of solubilizing IBs to improve protein recovery.

Solubilization of Human Interferon ß-1b Inclusion Body Proteins by Organic Solvents.

Purposes: Solubilization of inclusion bodies expressed in E. coli is a critical step during manufacturing of recombinant proteins expressed as inclusion bodies. So far, various methods have been used for solubilization and purification of inclusion body proteins to obtain active proteins with high purity and yield. The aim of this study was to examine the benefit of organic solvents such as alcohols in solubilization of recombinant interferon ß-1b inclusion bodies. Methods: Effect of important parameters inclusion pH, concentration and type of denaturant and concentration of alcoholic solvents were optimized to formulate a suitable solubilization buffer and investigate their effect on solubilization of interferon ß-1b inclusion bodies. Results: Our findings showed the acidic pH in the range of 2-3 is more suitable than alkaline pH >12 for solubilization and achieving higher content of interferon ß-1beta and pure recombinant protein. We have also demonstrated that 1% SDS acts better than 2M urea to solubilize Inclusion body proteins of interferon ß-1b at pH of 2-3. The interferon concentration was 2.35 mg per 100 mg IB when we used 40% (v/v) 1-propanol and 20% (v/v) 2-butanol into the buffer solution as well. Conclusion: The optimized method provides gentile condition for solubilization of inclusion body at high protein concentration and purity with a degree of retention of native secondary structure which makes this method valuable to be used in production and research area.

Promising insights into the kosmotropic effect of magnetic nanoparticles on proteins: The pivotal role of protein corona formation.

Increasing concerns about biosafety of nanoparticles (NPs) has raised the need for detailed knowledge of NP interactions with biological molecules especially proteins. Herein, the concentration-dependent effect of magnetic NPs (MNPs) on bovine serum albumin and hen egg white lysozyme was explored. The X-ray diffraction patterns, zeta potential, and dynamic light scattering measurements together with scanning electron microscopy images were employed to characterize MNPs synthesized through coprecipitation method. Then, we studied the behavior of two model proteins with different surface charges and structural properties on interaction with Fe3 O4 . A thorough investigation of protein-MNP interaction by the help of intrinsic fluorescence at different experimental conditions revealed that affinity of proteins for MNPs is strongly affected by the similarity of protein and MNP surface charges. MNPs exerted structure-making kosmotropic effect on both proteins under a concentration threshold; however, binding strength was found to determine the extent of stabilizing effect as well as magnitude of the concentration threshold. Circular dichroism spectra showed that proteins with less resistance to conformational deformations are more prone to secondary structure changes upon adsorption on MNPs. By screening thermal aggregation of proteins in the presence of Fe3 O4 , it was also found that like chemical stability, thermal stability is influenced to a higher extent in more strongly bound proteins. Overall, this report not only provides an integrated picture of protein-MNP interaction but also sheds light on the molecular mechanism underling this process.

Determination of Biological Activity of Recombinant Reteplase Using Clot Lysis Time and Activated Partial Thromboplastin Time (APTT) Lysis Methods: A Comparative Study.

Recombinant plasminogen activator (reteplase) is a third generation thrombolytic agent which has been used on coronary artery thrombosis and acute myocardial infarction. Clot lysis assay is usually considered as a unique method to evaluate biological activity of reteplase. In this study biological activity of reteplase was determined by APTT (activated partial thromboplastin time) lysis method. Validity of this method was evaluated in comparison with reference method, clot lysis time assay. Results of APTT lysis test showed good reproducibility (relative standard deviation (RSD) 3-5% for within day analysis and 4-7% for between day analysis), and accuracy (101.3-102.7%). APTT lysis responses were linear in range of 0.001-0.1 mg/mL reteplase. Therefore, APTT lysis method is applicable for biological activity determination of reteplase. Although more comprehensive studies are required to approve this test as a reference method, APTT lysis method seems to be valuable to receive more attention due to advantages of technical simplicity, sensitivity, applicability, and cost efficiency.

Effect of Nonionic Surfactants (Dodecyl Maltoside and Polysorbate 20) on Prevention of Aggregation and Conformational Changes of Recombinant Human IFNß_1b Induced by Light.

Liquid protein formulations are prone to form aggregates. The effect of nonionic surfactants such as Polysorbate 20 (PS 20) and n-Dodecyl ß-D-maltoside (DDM) on the prevention of aggregation and conformational changes of recombinant human IFNß-1b (rhIFN ß_1b) was explored. Polysorbate has been used in formulations of protein pharmaceuticals. There have been concerns about using PS 20 due to its residual peroxide content which may negatively affect protein efficacy. n-Dodecyl ß-D-maltoside has been of interest and shown to be highly effective in prevention of aggregation. Fresh bulk of rhIFN ß_1b was formulated using DDM or different concentrations of PS 20. Formulations were exposed to light stress condition according to the ICH guideline of Q1b. The overall conformational integrity of individual samples was characterized by a combination of Circular dichroism (CD), Fluorescence spectroscopy and RP_HPLC techniques. The CD spectrum depicting the conformational integrity of rhIFN ß_1b showed 31.9% and 31.2% decreases in α-helix content of protein samples with 0.2% or 0.02% of PS20 compared to only18.2% of that containing 0.2% DDM. The RP-HPLC analysis also showed that the oxidized impurity in formulation containing DDM is less than those contain PS 20. Complementary analysis of the liquid formulations using IFR and UV methods also was in compliance with the data obtained by CD. Compared to PS 20, the sample of rhIFN ß_1b formulation with DDM was more resistant to the destruction effect of light. Results were in accordance with previous studies and could suggest DDM as a reliable anti-aggregation surfactant in biopharmaceutical formulations.

Immunogenicity of Recombinant Human Interferon Beta-1b in Immune-Tolerant Transgenic Mice Corresponds with the Biophysical Characteristics of Aggregates.

Determining to what extent biophysical characteristics of aggregates affect immunogenicity of therapeutic interferon beta-1b. Three recombinant human interferon beta-1b (rhIFNß-1b) samples with different levels of aggregates generated by copper oxidation, thermal stress, or left untreated, as well as Avonex(®) drug substance and Betaferon(®) drug product, were injected intraperitoneally in nontransgenic and interferon beta transgenic FVB/N mice 5 times per week for 3 weeks. Antibodies against interferon beta were measured using enzyme-linked immunosorbent assay. UV and fluorescence spectroscopy, dynamic light scattering, size exclusion chromatography, reversed-phase high-performance liquid chromatography (RP-HPLC), fluid imaging microscopy, and resonant mass measurement, as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting, were used to characterize and quantitate aggregates in the 3 rhIFNß preparations, to correlate biophysical characteristics with immunogenicity. In immune-tolerant interferon beta transgenic FVB/N mice, Betaferon drug product showed the highest immunogenicity, while Avonex drug substance showed the lowest level of immunogenicity. Of the 3 forms of rhIFNß-1b, copper-oxidized rhIFNß-1b showed lower immunogenicity than thermally stressed rhIFNß-1b, despite containing larger aggregates. Both copper-oxidized rhIFNß-1b and thermally stressed rhIFNß-1b exhibited changes in protein structure as shown using fluorescence spectroscopy and RP-HPLC. Nontransgenic, nonimmune-tolerant FVB/N mice generated high antibody titers against all interferon beta samples tested. The level of immunogenicity and the breaking of tolerance in FVB/N transgenic mice are not only related to the level of aggregation but also depend on the size and structure of the aggregates.

An Albumin-Free Formulation for Escherichia coli-Derived Interferon Beta-1b with Decreased Immunogenicity in Immune Tolerant Mice.

Human serum albumin (HSA)-free formulation of Escherichia coli-derived human interferon beta (IFNß-1b) with a high percentage of monomeric protein and low immunogenicity is developed and characterized in the current study. UV spectroscopy, fluorescence spectroscopy, dynamic light scattering, sodium dodecyl sulfate polyacrylamide gel electrophoresis, Western blotting, Micro-Flow Imaging, resonant mass measurement, size exclusion, and reversed-phase high performance liquid chromatographies were applied to assess the effect of excipients on the stability of IFNß-1b to establish a HSA-free formulation. The antiviral activity of IFNß-1b was evaluated using human lung carcinoma cell line. Immune tolerant mice to hIFNß were used to assess the immunogenicity of the HSA-free formulated IFNß-1b in comparison to Betaferon drug product and Avonex drug substance as standards through IgG titering of plasma. HSA-free formulated IFNß-1b, including 200 mM L-arginine, 200 mM trehalose, and 0.1% n-dodecyl ß-D-maltoside in 10 mM sodium acetate buffer, pH 7.4, showed the highest biological activity. The stability of IFNß-1b in the HSA-free formulation was monitored for 3 weeks at 4°C and 37°C with relative humidity of 10% and 75%, respectively. Protein aggregation and immunogenicity in transgenic mice were decreased in the HSA-free formulated IFNß-1b compared to Betaferon. The stability, biological activity, and immunogenicity of the HSA-free formulation and Betaferon were evaluated. Incubation of formulations at 4°C and 37°C for 3 weeks showed that the HSA-free formulated IFNß-1b was more stable and less immunogenic in transgenic FVB/N mice. Low immunogenicity and the absence of HSA, which reduces the potential risk of viral infection (eg, HIV and HCV), are promising for clinical studies.

Preventing Aggregation of Recombinant Interferon beta-1b in Solution by Additives: Approach to an Albumin-Free Formulation.

PURPOSE: Aggregation suppressing additives have been used to stabilize proteins during manufacturing and storage. Interferonß-1b is prone to aggregation because of being non-glycosylated. Aggregation behavior of albumin-free formulations of recombinant IFNß-1b was explored using additives such as n-dodecyl-ß-D-maltoside, Tween 20, arginine, glycine, trehalose and sucrose at different pH. METHODS: Fractional factorial design was applied to select major factors affecting aggregation in solutions. Box-Behnken technique was used to optimize the best concentration of additives and protein. RESULTS: Quadratic model was the best fitted model for particle size, OD350 and OD280/OD260. The optimal conditions of 0.2% n-Dodecyl-ß-D-maltoside, 70 mM arginine, 189 mM trehalose and protein concentration of 0.50 mg/ml at pH 4 were achieved. A potency value of 91% ± 5% was obtained for the optimized formulation. CONCLUSION: This study shows that the combination of n-Dodecyl-ß-D-maltoside, arginine and trehalose would demonstrate a significant stabilizing and anti-aggregating effect on the liquid formulation of interferonß-1b. It can not only reduce the manufacturing costs but will also ease patient compliance.

The effects of dodecyl maltoside and sodium dodecyl sulfate surfactants on the stability and aggregation of recombinant interferon Beta-1b.

Aggregation often occurs during manufacturing and storage of protein drugs. Detergents such as sodium dodecyl sulfate are commonly used to prevent aggregation but need to be eliminated before final formulation for safety reasons. We studied the ability of dodecylmaltoside (DDM), a nontoxic alkyl saccharide surfactant, to reduce aggregation and increase the stability of interferon beta-1b (IFN)-ß-1b. An increase of 8°C in the Tm of IFN-ß-1b was observed when 0.1% of DDM was present in the protein solution. The absorption of DDM on hydrophobic surfaces of IFN-ß-1b enables the surface to become hydrophilic and non-ionic, and increases the stability of the protein. 0.1% DDM also results in a 62% increase in helical and a 25% decrease in ß-sheet structures. 0.1% DDM not only suppresses aggregate formation but also improves IFN-ß-1b solubilization. Furthermore, we have showed the protective effect of DDM on the anti-viral activity of IFN-ß-1b in solution.

The role of trehalose for metastable state and functional form of recombinant interferon beta-1b.

Physical and mathematical methods such as fluorescence and circular dichroism spectroscopies as well as MCR-ALS chemometric analysis and Wyman linkage theory were applied to assess the effect of trehalose on stability of refolded IFNß-1b (interferon beta-1b). An intermediate, showing native-like secondary structure is possibly formed during refolding process of IFNß-1b. Although the refolded protein showed high biological activity, the mathematical data demonstrated a flexible and mid-stable structure, possibly due to its non-glycosylated form. In the presence of 1.5 M of trehalose an increase of 16 °C was observed in the Tm of the refolded protein from the initial of 35 °C. The stability of IFNß-1b was possibly improved by preferential exclusion of trehalose and formation of a dense hydration shell around the protein surface. Lower amounts of helical structures (16%) were detected in protein solutions without trehalose. In the presence of trehalose the active structure of IFNß-1b was probably preserved by formation of a metastable structure which subsequently eased post-refolding processes of the protein.